American Journal of Transplantation 2015; XX: 1–6 Wiley Periodicals Inc.
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13300
Case Report
Transfer of HLA-Specific Allosensitization From a Highly Sensitized Deceased Organ Donor to the Recipients of Each Kidney S. J. Maxfield1, C. J. Taylor1,*, V. Kosmoliaptsis2, V. Broecker3, C. J. E. Watson2, J. A. Bradley2 and S. Peacock1
Received 19 December 2014, revised 05 February 2015 and accepted for publication 26 February 2015
1
Histocompatibility and Immunogenetics Laboratory, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK 2 Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, UK 3 Department of Histopathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK Corresponding author: Craig J. Taylor, [email protected]
We report for the first time the adoptive transfer of donor HLA-specific allosensitization in two recipients following kidney transplantation from a highly sensitized donor. Kidneys from a donation after circulatory death donor were transplanted into two nontransfused, HLA-specific antibody negative males receiving their first transplant. Antibody screening 7 days after transplant showed high level de novo IgG HLA class I- and class II-specific antibodies in both recipients, with largely overlapping antibody profiles but no antibodies to donor HLA. The unusually rapid appearance of de novo alloantibodies in immunosuppressed nonsensitized recipients and absence of donor HLA-specific antibody prompted testing of stored donor serum that revealed high antibody levels with specificities very similar to those seen in both recipients, but in addition the presence of strong antibodies to each recipient HLA. Alloantibody levels gradually declined but were still detectable at 3 months. These findings suggest that alloreactive passenger B cells/plasma cells within the kidneys of highly sensitized donors may give rise to rapid development of posttransplant de novo HLA-specific alloantibodies. While the clinical significance of this phenomenon is uncertain it provides one explanation for the appearance of de novo HLA-specific antibodies directed against third party but not donor HLA. Abbreviations: AMR, antibody-mediated rejection; DCD, donation after circulatory death; DSA, donor HLA-specific antibodies; HLA, human leukocyte antigens; HLA-SAB, single-antigen HLA-specific antibody detection beads; IgG, immunoglobulin G; MFI, median fluorescence intensity
Introduction Screening for the presence of HLA-specific antibodies after kidney transplantation is important for the diagnosis and management of antibody-mediated rejection (AMR) and has become routine in many transplant centers. The early appearance of de novo donor HLA-specific antibodies (DSA) after transplantation is of particular concern as acute antibody-mediated rejection (AMR) is often refractory to conventional anti-rejection therapy and is a major barrier to a successful transplant outcome (1,2). Although current immunotherapy has significantly reduced the incidence of acute T cell–mediated rejection, acute cellular rejection still occurs in around 10–20% of patients but is usually responsive to treatment, with minimal impact on future graft function. In contrast, early AMR in recipients receiving effective immunosuppression indicates a secondary immune response that is resistant to maintenance therapy and is not easily reversed (3). While DSA, either alone or in combination with microscopic evidence of microcirculation injury with or without C4d staining on kidney biopsy, are of most concern, the development of de novo nondonor HLA-specific (third party) antibodies are also, for reasons that are not clear, sometimes associated with an increased risk of graft loss (2,4–6). One possible explanation for the association between de novo third party HLA-specific antibodies and inferior transplant outcome is that third party antibodies occur in conjunction with low level DSA or other non–HLAspecific antibodies that are not detected in the circulation because they have been absorbed by the kidney graft (2). The development of posttransplant de novo DSA and third party HLA-specific antibodies can often be attributed to a memory response and are the end-product of an alloimmune response initiated by alloantigens expressed by the donor kidney that culminates in the production of antibody by recipient plasma cells. A previously unexplored possibility is that de novo HLA-specific antibodies 1
Maxfield et al
may also result from alloantibody producing passenger B lymphocytes/plasma cells present within the donor organ at the time of transplantation that after transfer into the immunosuppressed recipient continue to produce alloantibody. This situation would be analogous to the transfer of immune competent B cells in an ABO compatible donor organ that produce antibodies to recipient ABO blood group antigens and cause transplant associated hemolytic anemia (7,8). We describe herein, for the first time, the appearance of high level de novo IgG HLA class I- and class II-specific allosensitization in two nonsensitized recipients of a primary kidney transplant that we attribute to the transfer of donor passenger leukocytes from the same HLA highly sensitized donor. Case report Kidneys from a controlled donation after circulatory death (DCD) donor were transplanted locally into two first-time recipients, both of whom were HLA-specific antibody negative, nontransfused males. Donor and recipient demographic details and HLA types are shown in Table 1. The donor was a 48-year-old blood group A Rhesus-Dpositive male with alcoholic cirrhosis who suffered respiratory failure following variceal banding for haematemesis. The recipients were aged 54 and 52 years, and both were blood group A Rhesus-D-positive, with endstage renal failure caused by autosomal dominant polycystic kidney disease. Donor-recipient HLA-A, -B, -DR mismatch grades were 2.1.1 and 2.2.2, respectively. The donor
asystolic warm ischemia time was 10 min and kidney cold ischemia times were 18 h, 4 min, and 13 h, respectively. Recipient immunosuppression comprised basiliximab induction therapy (20 mg IV day 1 and day 3), and tacrolimus (Advagraf) with mycophenolate mofetil and prednisolone maintenance. Both recipients experienced delayed graft function that prompted clinically indicated kidney biopsy and donor HLA-specific antibody testing on day 7. Histological features in the biopsy from recipient 1 were consistent with diffuse acute tubular injury but no evidence of graft rejection, whereas the biopsy from recipient 2 showed diffuse acute tubular injury, interstitial oedema, focal interstitial haemorrhage, glomerular thrombi, and acute endothelialitis consistent with acute vascular rejection (Banff IIb) although staining for C4d was negative. This was treated successfully with pulsed steroids. Both recipients became dialysis-free and their serum creatinine levels were 1.80 mg/dL and 3.39 mg/dL, respectively, at three months. Day 7 posttransplant recipient serum samples were tested using Luminex HLA class I and class II antibody detection beads (LABScreen Mixed Bead MixTM, One Lambda, Canoga Park, CA) that showed surprisingly high level de novo HLA class I- and class II-specific antibodies (recipient 1 maximum bead ratio 50 and 211 for HLA class I and class II, respectively; recipient 2 maximum bead ratio 27 and 187 for HLA class I and class II, respectively). Further antibody characterization using Luminex single-antigen HLA-specific antibody detection beads (HLA-SAB) (LABScreen class I
Table 1: Donor and recipient demographics and HLA types Donor Primary disease Age and gender ABO-Rhesus D Warm ischemia time Cold ischemia time (hours:minutes) HLA-A, -B, -DR mismatch grade Serum creatinine day 7 (mg/dL) Serum creatinine day 90 (mg/dL) Pretransplant alloantibodies (cRF%)2 Day 7 posttransplant alloantibodies (cRF%) Day 90 posttransplant alloantibodies (cRF%) HLA-A* HLA-B* HLA-C* HLA-DRB1* HLA-DRB3-5* HLA-DQB1* HLA-DPB1*
— 48-year-old male A-positive 10 min — — — — 100% — — 32, 33 44, 51 07, 15 07, 10 4*01, — 02, 05 04, —
Recipient 1 1
ADPKD 54-year-old male A-positive — 18:04 2.1.1 9.48 1.80 Negative 99% 98% 02,033 44, 35 04, 05 01, 07 4*01, — 02, 05 04, 35
Recipient 2 ADPKD 52-year-old male A-positive — 13:00 2.2.2 7.14 3.39 Negative 98% 68% 23, 31 07, 49 07, — 15, — 5*01, — 06, — 04, —
1
ADPKD, autosomal dominant polycystic kidney disease. Calculated reaction frequency (cRF%): the HLA antibody specificities identified in sera using Luminex single-antigen beads (MFI >500) were used to determine the cRF. cRF defines the percentage of a standardized panel of 10 000 consecutive HLA-typed UK deceased organ donors (considered as representative of HLA types in the UK population) that is incompatible with the alloantibody profile of a given patient [Howell WM et al Int. J. Immunogenetics 2010; 37: 435–437]. 3 Recipient HLA types shown in bold highlight specificities to which the donor had high level recipient-HLA-specific alloantibodies (MFI >5000). 2
2
American Journal of Transplantation 2015; XX: 1–6
Donor Derived De Novo HLA-Allosensitization
and class II single-antigen beadsTM, One Lambda) identified high level antibody binding in both recipient sera with largely overlapping antibody profiles (Fisher’s exact probability of association p < 0.000001). The mean fluorescent intensity (MFI) against individual HLA-SAB populations was higher in recipient 1 but neither recipient had detectable antibodies to the donor HLA type. Analysis of day 7 sera showed that of the 40 antibody-positive (MFI >500) HLA-SAB populations identified in recipient 1 and the 32 antibody-positive HLA-SAB populations in recipient 2, 19 were identical. Of the 34 HLA-SAB alloantibody specificities not shared between recipient 1 and recipient 2, 20 (59%) were directed to their own HLA type or closely related HLA specificities that share serological cross-reactive epitopes with the recipient HLA type. The appearance of such strong and very similar HLAspecific antibody profiles in two nonsensitized immunosuppressed recipients within one week of a primary transplant is unusual and this, together with the absence of DSA prompted screening of archived donor serum obtained prior to organ retrieval. Donor serum HLA-SAB screening results identified that the donor was highly sensitized and had a largely overlapping antibody profile to that seen in both recipients’ posttransplant serum (donor vs. recipient 1 Fisher’s exact probability of association p < 0.000001: donor vs. recipient 2 Fisher’s exact probability of association p ¼ 0.001), but in addition, contained high levels of alloantibody to HLA specificities expressed by each of the two recipients. Of the 131 antibody-positive HLA-SAB populations identified in donor serum, 40 were detected in sera from recipient 1 and 29 were detected in sera from recipient 2 (Table 2). All antibody positive HLASAB populations antibody specificities in serum from recipient 1 and all except three specificities detected in recipient 2 were present in the donor serum (all three additional specificities present in recipient 2 but not the donor had borderline values close to the 500 MFI cut-off threshold). Of the 193 antibody-positive HLA-SAB
populations identified using donor serum that were absent with recipient 1 (N ¼ 91) or recipient 2 (N ¼ 102) serum, 40 and 32 respectively were accounted for by recipients’ own HLA types or by antibodies against closely related HLA specificities that share serological epitopes with the recipient HLA type (Table 2). Taken together, these results indicate that the posttransplant HLA-specific antibodies detected in both recipients were not caused by de novo recipient sensitization, but instead were acquired from donor-derived antibody-producing cells. Interestingly, donor alloantibodies also included high-level recipient HLA-specific antibodies, but these were negative in the respective recipient serum, whereas third party antibodies (specific to neither donor nor recipient HLA) were present in donor and both recipient sera (Table 3). Figure 1 shows the antibody MFI values over time: Recipient 1 had high-level antibody binding (MFI >10 000) to multiple third-party HLA-specificities that remained largely unchanged until beyond day 40 and slowly declined thereafter, but were still present at 5 months. Recipient 2 had intermediate level antibody binding detected on day 7 that declined rapidly until day 50, and were present at low MFI levels at day 90.
Discussion The two cases described in this report suggest that adoptive transfer of alloreactive passenger B cells/plasma cells present within the kidneys of a highly sensitized donor may give rise to the rapid development of posttransplant de novo HLA-specific alloantibodies directed against third party HLA. Serum from the sensitized donor showed strong HLA-specific antibodies to each of the recipient HLA types, but since recipient HLA-specific antibodies were not detected in recipient sera, we assume they were either absorbed by recipient tissues or alloantigen-specific modulation of autoreactive clones occurred. If the former
Table 2: Analysis of preretrieval donor serum and day 7 posttransplant recipient serum antibody binding to corresponding HLA class I and class II single-antigen antibody detection beads (MFI >500)
Number of HLA antibody specificities in donor serum identified in recipient serum Number of HLA antibody specificities in donor serum but absent in recipient serum Number of HLA antibody specificities in recipient serum but absent in donor serum Number of HLA antibody specificities absent in both donor and recipient serum Total number of single antigen bead specificities Chi-square p-value
Recipient 1
Recipient 2
40
29
911
1022
0
3
61
58
192 22 0.000003
192 7.69 0.006
1
Of the 91 single-antigen bead populations that were positive with donor serum but negative with recipient 1.40 (44%) were self-HLA or cross-reactive (sharing a common epitope) with the recipient HLA type. 2 Of the 102 single-antigen bead populations that were positive with donor serum but negative with recipient 2.32 (31%) were self-HLA or cross-reactive (sharing a common epitope) with the recipient HLA type.
American Journal of Transplantation 2015; XX: 1–6
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Maxfield et al Table 3: HLA-specific antibodies identified in kidney donor serum obtained during organ retrieval and recipient 1 and recipient 2 sera obtained on day 7 and 3 months posttransplant HLAspecificity A2 A68 A69 A3 A11 DR1 A23 A31 A30 B7 Cw7 DR15 DR16 DR51 DQ6 A1 A24 DR17 DR8 DR11 DR12 DR13 DR14
Donor antibody (MFI)
Recipient 1
Recipient 1 Day 7 (MFI)
Recipient 1 Day 112 (MFI)
Recipient 2
Recipient 2 Day 7 (MFI)
Recipient 2 Day 95 (MFI)
14 300 15 896 14 601 16 779 15 175 14 677 15 364 5837 7411 3796 3399 20 060 19 597 19 723 7475 12 365 11 889 18 504 19 365 18 373 20 212 17 672 20 359
self-HLA1 self-CREG self-CREG self-HLA self-CREG self-HLA third party third party third party third party third party third party third party third party third party third party third party third party third party third party third party third party third party
24 0 0 8 77 2 4738 400 0 29 0 17 275 15 034 2265 0 6351 4631 18 700 17 685 16 302 14 838 18 278 18 942
49 25 0 1 0 21 811 317 35 38 0 9684 7518 233 0 1208 894 13 860 14 142 12 201 10 433 16 531 15 029
third party third party third party third party third party third party self-HLA self-HLA self-CREG self-HLA self-HLA self-HLA self-CREG self-HLA self-HLA third party third party third party third party third party third party third party third party
1666 902 1435 1953 1802 41 26 0 0 47 0 48 89 0 0 4166 1514 10 057 10 128 7232 5002 9915 9631
238 82 140 245 191 7 0 0 0 2 0 0 0 0 0 1103 174 900 697 487 358 908 810
1
The HLA-antibody specificities show antigen-specific absorption/modulation against self-HLA (recipient own HLA type) and closely related cross-reactive epitopes (self-CREG), and examples of high-level third party antibodies that are not related to the recipient HLA type.
explanation is responsible, antibody absorption by recipient tissue was not accompanied by any obvious short-term pathological effects such as transfusion-related acute lung injury or other forms of graft-versus-host disease in either of the recipients. Luminex technology is highly sensitive and the level and duration of antibody that is required to cause autoimmune syndromes, transfusion-related acute lung injury or organ allograft rejection is probably much higher than that which might have been produced by transfer of donor plasma cells into a transplant recipient. It is notable, for example, that recipients of lung or liver transplants that develop high levels of donor HLA-specific antibody do not necessarily show acute lung or liver injury. The observation that third party antibodies were still detected beyond 5 months, albeit reduced in level from that early after transplantation, suggests that donor-derived alloantibody producing plasma cells persist in the recipient for at least several months, since the half-life of IgG is only around 7–23 days. Nearly all the HLA antibodies specificities identified in the two recipients were present in donor serum, but in addition the donor had antibodies to many HLA specificities that were not subsequently identified in posttransplant sera from either of the two recipients. There are three potential explanations for this. First, a number of HLA-specific antibodies identified in donor serum were only present at 4
low MFI levels and these accounted for much of the discrepancy in antibody profiles. Second, not all antibodyproducing B cell/plasma cell clones from the donor may have been adoptively transferred to the recipients. Third, our analysis using common serological cross-reactive groups likely provides an underestimate of the number of shared epitopes expressed on recipient HLA that absorb alloantibodies produced by transferred donor B cells/ plasma cells. Although the present report is the first to suggest transfer of HLA-specific allosensitization from a highly sensitized organ donor, passenger lymphocyte syndrome is a well recognized, but rare, complication following solid organ transplantation that differs in frequency and clinical manifestation according to organ type (9). It occurs when primed immunecompetent donor cells present within the graft are transferred into an immune compromised recipient. The most common manifestation is the antibody-mediated hemolytic anemia that follows ABO-compatible but nonidentical transplantation mediated by donor-derived B cells/ plasma cells primed to host allogeneic red blood cell antigens (7,8,10,11). Other manifestations may include acute graftversus-host disease mediated by donor-derived alloreactive cytotoxic T cells that attack host gut, skin, bone marrow, liver, and lungs (12). Other examples of recipient acquired cellular and/or antibody immunity caused by concomitant transfer of donor lymphocytes following solid organ American Journal of Transplantation 2015; XX: 1–6
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A
22,000 20,000 18,000 16,000
MFI
14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
Days posransplant
B
12,000
10,000
MFI
8,000
6,000
4,000
2,000
0 0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
Days posransplant Figure 1: Posttransplant antibody binding (median fluorescent intensity; MFI) to Luminex single-antigen HLA-class I and class II antibody detection beads. Recipient 1 (A) had high-level antibody binding (MFI >10 000) to multiple third-party HLA-specificities (negative for donor and recipient HLA) detected on day 7 that remained largely unchanged until beyond day 40, and slowly declined thereafter. Recipient 2 (B) had moderate-level antibody binding detected on day 7 (with largely similar antibody binding profile to recipient 1) that declined rapidly until day 50, and was present at low MFI levels beyond day 90.
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transplantation include idiopathic thrombocytopenia (13), vitiligo (14), and peanut allergy (15). In patients with graftversus-host disease after liver, lung, or intestinal transplantation donor T cell chimerism is well described (12), but is very rare after kidney or heart transplantation. Donor B cell microchimerism has, however, been detected in patients who developed red blood cell alloantibodies following liver and kidney–pancreas transplantation (16). In the two recipients described here, the novel suggestion that the development of posttransplant de novo HLA-specific alloantibodies was caused by the adoptive transfer of donor alloreactive passenger B cells/plasma cells within the kidney was not initially apparent and we did not therefore test for peripheral blood donor B cell chimerism. The clinical significance of the transfer of donor HLA-specific allosensitization described in this report is uncertain but there were no overt short-term clinical manifestations in either of the two recipients. The phenomenon described provides one possible explanation for the observation of high level de novo HLA-specific antibodies against third party HLA antigens following kidney transplantation.
Acknowledgments CJT, VK, and JAB are supported by the NIHR Cambridge Biomedical Research Center.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
References €sal C. Clinical relevance of HLA 1. Morath C, Opelz G, Zeier M, Su antibody monitoring after kidney transplantation. J Immunol Res 2014. doi: 10.1155/2014/845040 2. Wiebe C, Gibson IW, Blydt-Hansen TD, et al. Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant. Am J Transplant 2012; 12: 1157–1167.
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3. Dunn TB, Noreen H, Gillingham K, et al. Revisiting traditional risk factors for rejection and graft loss after kidney transplantation. Am J Transplant 2011; 11: 2132–2143. 4. Ntokou ISA, Iniotaki AG, Kontou EN, et al. Long-term follow up for anti-HLA donor specific antibodies post renal transplantation: High immunogenicity of HLA class II graft molecules. Transpl Int 2011; 24: 1084–1093. 5. Hourmant M, Cesbron-Gautier A, Terasaki PI, et al. Frequency and clinical implications of development of donor-specific and nondonor-specific HLA antibodies after kidney transplantation. J Am Soc Nephrol 2005; 16: 2804–2812. 6. Lachmann N, Terasaki PI, Budde K, et al. Anti-human leukocyte antigen and donor-specific antibodies detected by luminex posttransplant serve as biomarkers for chronic rejection of renal allografts. Transplantation 2009; 87: 1505–1513. 7. Mangal AK, Growe GH, Sinclair M, Stillwell GF, Reeve CE, Naiman SC. Acquired hemolytic anemia due to ‘‘auto’’-anti-A or ’’auto’’anti-B induced by group O homograft in renal transplant recipients. Transfusion 1984; 24: 201–205. 8. Orchard J, Young NT, Smith C, Thomas S, Darke C. Severe intravascular haemolysis in a renal transplant recipient due to anti-B of donor origin. Vox Sanguinis 1990; 59: 172–175. 9. Zhang Y, Ruiz P. Solid organ transplant-associated acute graftversus-host disease. Arch Pathol Lab Med 2010; 134: 1220–1224. 10. Ainsworth CD, Crowther MA, Treleaven D, Evanovitch D, Webert KE, Blajchman MA. Severe hemolytic anemia post-renal transplantation produced by donor anti-D passenger lymphocytes: Case report and literature review. Transfus Med Rev 2009; 23: 155–159. 11. Bakr MA, Abbas TM, Mustafa A, Ghoneim MA. Hemolytic anemia after ABO non-identical living donor kidney transplantation. Clin Exp Nephrol 2009; 13: 161–165. 12. Taylor AL, Gibbs P, Sudhindran S, et al. Monitoring systemic donor lymphocyte macrochimerism to aid the diagnosis of graft-versushost disease after liver transplantation. Transplantation 2004; 77: 441–446. 13. Pereboom ITA, De Boer MT, Haagsma EB, et al. Transmission of idiopathic thrombocytopenic purpura during orthotopic liver transplantation. Transpl Int 2010; 23: 236–238. 14. Bradley V, Kemp EH, Dickinson C, Key T, Gibbs P, Clatworthy MR. Vitiligo following a combined liver-kidney transplant. Nephrol Dial Transplant 2009; 24: 686–688. 15. Bhinder S, Heffer MJ, Lee JK, Chaparro C, Tarlo SM. Development of transient peanut allergy following lung transplantation: A case report. Can Respir J 2011; 18: 154–156. 16. Seltsam A, Hell A, Heymann G, Salama A. Donor-derived alloantibodies and passenger lymphocyte syndrome in two of four patients who received different organs from the same donor. Transfusion 2001; 41: 365–370.
American Journal of Transplantation 2015; XX: 1–6
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Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: 10.1111/ajt.13300
Case Report
Transfer of HLA-Specific Allosensitization From a Highly Sensitized Deceased Organ Donor to the Recipients of Each Kidney S. J. Maxfield1, C. J. Taylor1,*, V. Kosmoliaptsis2, V. Broecker3, C. J. E. Watson2, J. A. Bradley2 and S. Peacock1
Received 19 December 2014, revised 05 February 2015 and accepted for publication 26 February 2015
1
Histocompatibility and Immunogenetics Laboratory, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK 2 Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Cambridge, UK 3 Department of Histopathology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK Corresponding author: Craig J. Taylor, [email protected]
We report for the first time the adoptive transfer of donor HLA-specific allosensitization in two recipients following kidney transplantation from a highly sensitized donor. Kidneys from a donation after circulatory death donor were transplanted into two nontransfused, HLA-specific antibody negative males receiving their first transplant. Antibody screening 7 days after transplant showed high level de novo IgG HLA class I- and class II-specific antibodies in both recipients, with largely overlapping antibody profiles but no antibodies to donor HLA. The unusually rapid appearance of de novo alloantibodies in immunosuppressed nonsensitized recipients and absence of donor HLA-specific antibody prompted testing of stored donor serum that revealed high antibody levels with specificities very similar to those seen in both recipients, but in addition the presence of strong antibodies to each recipient HLA. Alloantibody levels gradually declined but were still detectable at 3 months. These findings suggest that alloreactive passenger B cells/plasma cells within the kidneys of highly sensitized donors may give rise to rapid development of posttransplant de novo HLA-specific alloantibodies. While the clinical significance of this phenomenon is uncertain it provides one explanation for the appearance of de novo HLA-specific antibodies directed against third party but not donor HLA. Abbreviations: AMR, antibody-mediated rejection; DCD, donation after circulatory death; DSA, donor HLA-specific antibodies; HLA, human leukocyte antigens; HLA-SAB, single-antigen HLA-specific antibody detection beads; IgG, immunoglobulin G; MFI, median fluorescence intensity
Introduction Screening for the presence of HLA-specific antibodies after kidney transplantation is important for the diagnosis and management of antibody-mediated rejection (AMR) and has become routine in many transplant centers. The early appearance of de novo donor HLA-specific antibodies (DSA) after transplantation is of particular concern as acute antibody-mediated rejection (AMR) is often refractory to conventional anti-rejection therapy and is a major barrier to a successful transplant outcome (1,2). Although current immunotherapy has significantly reduced the incidence of acute T cell–mediated rejection, acute cellular rejection still occurs in around 10–20% of patients but is usually responsive to treatment, with minimal impact on future graft function. In contrast, early AMR in recipients receiving effective immunosuppression indicates a secondary immune response that is resistant to maintenance therapy and is not easily reversed (3). While DSA, either alone or in combination with microscopic evidence of microcirculation injury with or without C4d staining on kidney biopsy, are of most concern, the development of de novo nondonor HLA-specific (third party) antibodies are also, for reasons that are not clear, sometimes associated with an increased risk of graft loss (2,4–6). One possible explanation for the association between de novo third party HLA-specific antibodies and inferior transplant outcome is that third party antibodies occur in conjunction with low level DSA or other non–HLAspecific antibodies that are not detected in the circulation because they have been absorbed by the kidney graft (2). The development of posttransplant de novo DSA and third party HLA-specific antibodies can often be attributed to a memory response and are the end-product of an alloimmune response initiated by alloantigens expressed by the donor kidney that culminates in the production of antibody by recipient plasma cells. A previously unexplored possibility is that de novo HLA-specific antibodies 1
Maxfield et al
may also result from alloantibody producing passenger B lymphocytes/plasma cells present within the donor organ at the time of transplantation that after transfer into the immunosuppressed recipient continue to produce alloantibody. This situation would be analogous to the transfer of immune competent B cells in an ABO compatible donor organ that produce antibodies to recipient ABO blood group antigens and cause transplant associated hemolytic anemia (7,8). We describe herein, for the first time, the appearance of high level de novo IgG HLA class I- and class II-specific allosensitization in two nonsensitized recipients of a primary kidney transplant that we attribute to the transfer of donor passenger leukocytes from the same HLA highly sensitized donor. Case report Kidneys from a controlled donation after circulatory death (DCD) donor were transplanted locally into two first-time recipients, both of whom were HLA-specific antibody negative, nontransfused males. Donor and recipient demographic details and HLA types are shown in Table 1. The donor was a 48-year-old blood group A Rhesus-Dpositive male with alcoholic cirrhosis who suffered respiratory failure following variceal banding for haematemesis. The recipients were aged 54 and 52 years, and both were blood group A Rhesus-D-positive, with endstage renal failure caused by autosomal dominant polycystic kidney disease. Donor-recipient HLA-A, -B, -DR mismatch grades were 2.1.1 and 2.2.2, respectively. The donor
asystolic warm ischemia time was 10 min and kidney cold ischemia times were 18 h, 4 min, and 13 h, respectively. Recipient immunosuppression comprised basiliximab induction therapy (20 mg IV day 1 and day 3), and tacrolimus (Advagraf) with mycophenolate mofetil and prednisolone maintenance. Both recipients experienced delayed graft function that prompted clinically indicated kidney biopsy and donor HLA-specific antibody testing on day 7. Histological features in the biopsy from recipient 1 were consistent with diffuse acute tubular injury but no evidence of graft rejection, whereas the biopsy from recipient 2 showed diffuse acute tubular injury, interstitial oedema, focal interstitial haemorrhage, glomerular thrombi, and acute endothelialitis consistent with acute vascular rejection (Banff IIb) although staining for C4d was negative. This was treated successfully with pulsed steroids. Both recipients became dialysis-free and their serum creatinine levels were 1.80 mg/dL and 3.39 mg/dL, respectively, at three months. Day 7 posttransplant recipient serum samples were tested using Luminex HLA class I and class II antibody detection beads (LABScreen Mixed Bead MixTM, One Lambda, Canoga Park, CA) that showed surprisingly high level de novo HLA class I- and class II-specific antibodies (recipient 1 maximum bead ratio 50 and 211 for HLA class I and class II, respectively; recipient 2 maximum bead ratio 27 and 187 for HLA class I and class II, respectively). Further antibody characterization using Luminex single-antigen HLA-specific antibody detection beads (HLA-SAB) (LABScreen class I
Table 1: Donor and recipient demographics and HLA types Donor Primary disease Age and gender ABO-Rhesus D Warm ischemia time Cold ischemia time (hours:minutes) HLA-A, -B, -DR mismatch grade Serum creatinine day 7 (mg/dL) Serum creatinine day 90 (mg/dL) Pretransplant alloantibodies (cRF%)2 Day 7 posttransplant alloantibodies (cRF%) Day 90 posttransplant alloantibodies (cRF%) HLA-A* HLA-B* HLA-C* HLA-DRB1* HLA-DRB3-5* HLA-DQB1* HLA-DPB1*
— 48-year-old male A-positive 10 min — — — — 100% — — 32, 33 44, 51 07, 15 07, 10 4*01, — 02, 05 04, —
Recipient 1 1
ADPKD 54-year-old male A-positive — 18:04 2.1.1 9.48 1.80 Negative 99% 98% 02,033 44, 35 04, 05 01, 07 4*01, — 02, 05 04, 35
Recipient 2 ADPKD 52-year-old male A-positive — 13:00 2.2.2 7.14 3.39 Negative 98% 68% 23, 31 07, 49 07, — 15, — 5*01, — 06, — 04, —
1
ADPKD, autosomal dominant polycystic kidney disease. Calculated reaction frequency (cRF%): the HLA antibody specificities identified in sera using Luminex single-antigen beads (MFI >500) were used to determine the cRF. cRF defines the percentage of a standardized panel of 10 000 consecutive HLA-typed UK deceased organ donors (considered as representative of HLA types in the UK population) that is incompatible with the alloantibody profile of a given patient [Howell WM et al Int. J. Immunogenetics 2010; 37: 435–437]. 3 Recipient HLA types shown in bold highlight specificities to which the donor had high level recipient-HLA-specific alloantibodies (MFI >5000). 2
2
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Donor Derived De Novo HLA-Allosensitization
and class II single-antigen beadsTM, One Lambda) identified high level antibody binding in both recipient sera with largely overlapping antibody profiles (Fisher’s exact probability of association p < 0.000001). The mean fluorescent intensity (MFI) against individual HLA-SAB populations was higher in recipient 1 but neither recipient had detectable antibodies to the donor HLA type. Analysis of day 7 sera showed that of the 40 antibody-positive (MFI >500) HLA-SAB populations identified in recipient 1 and the 32 antibody-positive HLA-SAB populations in recipient 2, 19 were identical. Of the 34 HLA-SAB alloantibody specificities not shared between recipient 1 and recipient 2, 20 (59%) were directed to their own HLA type or closely related HLA specificities that share serological cross-reactive epitopes with the recipient HLA type. The appearance of such strong and very similar HLAspecific antibody profiles in two nonsensitized immunosuppressed recipients within one week of a primary transplant is unusual and this, together with the absence of DSA prompted screening of archived donor serum obtained prior to organ retrieval. Donor serum HLA-SAB screening results identified that the donor was highly sensitized and had a largely overlapping antibody profile to that seen in both recipients’ posttransplant serum (donor vs. recipient 1 Fisher’s exact probability of association p < 0.000001: donor vs. recipient 2 Fisher’s exact probability of association p ¼ 0.001), but in addition, contained high levels of alloantibody to HLA specificities expressed by each of the two recipients. Of the 131 antibody-positive HLA-SAB populations identified in donor serum, 40 were detected in sera from recipient 1 and 29 were detected in sera from recipient 2 (Table 2). All antibody positive HLASAB populations antibody specificities in serum from recipient 1 and all except three specificities detected in recipient 2 were present in the donor serum (all three additional specificities present in recipient 2 but not the donor had borderline values close to the 500 MFI cut-off threshold). Of the 193 antibody-positive HLA-SAB
populations identified using donor serum that were absent with recipient 1 (N ¼ 91) or recipient 2 (N ¼ 102) serum, 40 and 32 respectively were accounted for by recipients’ own HLA types or by antibodies against closely related HLA specificities that share serological epitopes with the recipient HLA type (Table 2). Taken together, these results indicate that the posttransplant HLA-specific antibodies detected in both recipients were not caused by de novo recipient sensitization, but instead were acquired from donor-derived antibody-producing cells. Interestingly, donor alloantibodies also included high-level recipient HLA-specific antibodies, but these were negative in the respective recipient serum, whereas third party antibodies (specific to neither donor nor recipient HLA) were present in donor and both recipient sera (Table 3). Figure 1 shows the antibody MFI values over time: Recipient 1 had high-level antibody binding (MFI >10 000) to multiple third-party HLA-specificities that remained largely unchanged until beyond day 40 and slowly declined thereafter, but were still present at 5 months. Recipient 2 had intermediate level antibody binding detected on day 7 that declined rapidly until day 50, and were present at low MFI levels at day 90.
Discussion The two cases described in this report suggest that adoptive transfer of alloreactive passenger B cells/plasma cells present within the kidneys of a highly sensitized donor may give rise to the rapid development of posttransplant de novo HLA-specific alloantibodies directed against third party HLA. Serum from the sensitized donor showed strong HLA-specific antibodies to each of the recipient HLA types, but since recipient HLA-specific antibodies were not detected in recipient sera, we assume they were either absorbed by recipient tissues or alloantigen-specific modulation of autoreactive clones occurred. If the former
Table 2: Analysis of preretrieval donor serum and day 7 posttransplant recipient serum antibody binding to corresponding HLA class I and class II single-antigen antibody detection beads (MFI >500)
Number of HLA antibody specificities in donor serum identified in recipient serum Number of HLA antibody specificities in donor serum but absent in recipient serum Number of HLA antibody specificities in recipient serum but absent in donor serum Number of HLA antibody specificities absent in both donor and recipient serum Total number of single antigen bead specificities Chi-square p-value
Recipient 1
Recipient 2
40
29
911
1022
0
3
61
58
192 22 0.000003
192 7.69 0.006
1
Of the 91 single-antigen bead populations that were positive with donor serum but negative with recipient 1.40 (44%) were self-HLA or cross-reactive (sharing a common epitope) with the recipient HLA type. 2 Of the 102 single-antigen bead populations that were positive with donor serum but negative with recipient 2.32 (31%) were self-HLA or cross-reactive (sharing a common epitope) with the recipient HLA type.
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Maxfield et al Table 3: HLA-specific antibodies identified in kidney donor serum obtained during organ retrieval and recipient 1 and recipient 2 sera obtained on day 7 and 3 months posttransplant HLAspecificity A2 A68 A69 A3 A11 DR1 A23 A31 A30 B7 Cw7 DR15 DR16 DR51 DQ6 A1 A24 DR17 DR8 DR11 DR12 DR13 DR14
Donor antibody (MFI)
Recipient 1
Recipient 1 Day 7 (MFI)
Recipient 1 Day 112 (MFI)
Recipient 2
Recipient 2 Day 7 (MFI)
Recipient 2 Day 95 (MFI)
14 300 15 896 14 601 16 779 15 175 14 677 15 364 5837 7411 3796 3399 20 060 19 597 19 723 7475 12 365 11 889 18 504 19 365 18 373 20 212 17 672 20 359
self-HLA1 self-CREG self-CREG self-HLA self-CREG self-HLA third party third party third party third party third party third party third party third party third party third party third party third party third party third party third party third party third party
24 0 0 8 77 2 4738 400 0 29 0 17 275 15 034 2265 0 6351 4631 18 700 17 685 16 302 14 838 18 278 18 942
49 25 0 1 0 21 811 317 35 38 0 9684 7518 233 0 1208 894 13 860 14 142 12 201 10 433 16 531 15 029
third party third party third party third party third party third party self-HLA self-HLA self-CREG self-HLA self-HLA self-HLA self-CREG self-HLA self-HLA third party third party third party third party third party third party third party third party
1666 902 1435 1953 1802 41 26 0 0 47 0 48 89 0 0 4166 1514 10 057 10 128 7232 5002 9915 9631
238 82 140 245 191 7 0 0 0 2 0 0 0 0 0 1103 174 900 697 487 358 908 810
1
The HLA-antibody specificities show antigen-specific absorption/modulation against self-HLA (recipient own HLA type) and closely related cross-reactive epitopes (self-CREG), and examples of high-level third party antibodies that are not related to the recipient HLA type.
explanation is responsible, antibody absorption by recipient tissue was not accompanied by any obvious short-term pathological effects such as transfusion-related acute lung injury or other forms of graft-versus-host disease in either of the recipients. Luminex technology is highly sensitive and the level and duration of antibody that is required to cause autoimmune syndromes, transfusion-related acute lung injury or organ allograft rejection is probably much higher than that which might have been produced by transfer of donor plasma cells into a transplant recipient. It is notable, for example, that recipients of lung or liver transplants that develop high levels of donor HLA-specific antibody do not necessarily show acute lung or liver injury. The observation that third party antibodies were still detected beyond 5 months, albeit reduced in level from that early after transplantation, suggests that donor-derived alloantibody producing plasma cells persist in the recipient for at least several months, since the half-life of IgG is only around 7–23 days. Nearly all the HLA antibodies specificities identified in the two recipients were present in donor serum, but in addition the donor had antibodies to many HLA specificities that were not subsequently identified in posttransplant sera from either of the two recipients. There are three potential explanations for this. First, a number of HLA-specific antibodies identified in donor serum were only present at 4
low MFI levels and these accounted for much of the discrepancy in antibody profiles. Second, not all antibodyproducing B cell/plasma cell clones from the donor may have been adoptively transferred to the recipients. Third, our analysis using common serological cross-reactive groups likely provides an underestimate of the number of shared epitopes expressed on recipient HLA that absorb alloantibodies produced by transferred donor B cells/ plasma cells. Although the present report is the first to suggest transfer of HLA-specific allosensitization from a highly sensitized organ donor, passenger lymphocyte syndrome is a well recognized, but rare, complication following solid organ transplantation that differs in frequency and clinical manifestation according to organ type (9). It occurs when primed immunecompetent donor cells present within the graft are transferred into an immune compromised recipient. The most common manifestation is the antibody-mediated hemolytic anemia that follows ABO-compatible but nonidentical transplantation mediated by donor-derived B cells/ plasma cells primed to host allogeneic red blood cell antigens (7,8,10,11). Other manifestations may include acute graftversus-host disease mediated by donor-derived alloreactive cytotoxic T cells that attack host gut, skin, bone marrow, liver, and lungs (12). Other examples of recipient acquired cellular and/or antibody immunity caused by concomitant transfer of donor lymphocytes following solid organ American Journal of Transplantation 2015; XX: 1–6
Donor Derived De Novo HLA-Allosensitization
A
22,000 20,000 18,000 16,000
MFI
14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
Days posransplant
B
12,000
10,000
MFI
8,000
6,000
4,000
2,000
0 0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
Days posransplant Figure 1: Posttransplant antibody binding (median fluorescent intensity; MFI) to Luminex single-antigen HLA-class I and class II antibody detection beads. Recipient 1 (A) had high-level antibody binding (MFI >10 000) to multiple third-party HLA-specificities (negative for donor and recipient HLA) detected on day 7 that remained largely unchanged until beyond day 40, and slowly declined thereafter. Recipient 2 (B) had moderate-level antibody binding detected on day 7 (with largely similar antibody binding profile to recipient 1) that declined rapidly until day 50, and was present at low MFI levels beyond day 90.
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Maxfield et al
transplantation include idiopathic thrombocytopenia (13), vitiligo (14), and peanut allergy (15). In patients with graftversus-host disease after liver, lung, or intestinal transplantation donor T cell chimerism is well described (12), but is very rare after kidney or heart transplantation. Donor B cell microchimerism has, however, been detected in patients who developed red blood cell alloantibodies following liver and kidney–pancreas transplantation (16). In the two recipients described here, the novel suggestion that the development of posttransplant de novo HLA-specific alloantibodies was caused by the adoptive transfer of donor alloreactive passenger B cells/plasma cells within the kidney was not initially apparent and we did not therefore test for peripheral blood donor B cell chimerism. The clinical significance of the transfer of donor HLA-specific allosensitization described in this report is uncertain but there were no overt short-term clinical manifestations in either of the two recipients. The phenomenon described provides one possible explanation for the observation of high level de novo HLA-specific antibodies against third party HLA antigens following kidney transplantation.
Acknowledgments CJT, VK, and JAB are supported by the NIHR Cambridge Biomedical Research Center.
Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.
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